During the last 5-7 years, we identified several new proteins that are components of novel G protein signaling pathways. The characterization of these proteins has brought to light new roles for trimeric G proteins in cell proliferation, cell migration, and mitochondrial dynamics. Our accomplishments during the last 5 years of this grant include: 1) Discovery of a novel regulatory role for G1s in EGF receptor (EGFR) downregulation and signaling; 2) Demonstration of the crucial role played by GIPC in TrkA NGF signaling and MAPK activation; 3) Identification of GIV, a novel G1 binding protein, and demonstration that activation of G1i3 and its interaction with GIV is essential for cell migration and Akt activation during cell migration; 4) Discovery of hNOA1 (formerly called MIG78), a new dynamin-like GTPase which may provide a link between mitochondrial dynamics, respiration and apoptosis; 5) Identification of MIR16 (GDE1), a novel enzyme that provides a putative link between phosphoinositide metabolism and G protein signaling; and 6) Discovery of a new paradigm for spatial regulation of G1i signaling in clathrin-coated membrane microdomains. The overall goal of the work proposed is to further define two of these novel pathways we have discovered-namely, the role of G1i3 and GIV in cell migration and Akt signaling and the role of G1s in regulation of EGFR trafficking and signaling. We will focus on three specific aims: Specific Aim #1: To assess the functional interaction between G1i3 and GIV during cell migration. Our working model is that GIV serves as a non-receptor GEF and is the trigger that activates the signaling cascade culminating in efficient cell motility. Specific Aim #2: To investigate the spatial and temporal regulation of G1i3 and GIV's interaction and translocation from the Golgi to the PM in migrating cells. We will investigate where they interact and where G1i3 is activated using fluorescent biosensors, FRET and FRAP in living cells. Specific Aim #3: To pinpoint the mechanisms by which G1s and RGS-PX1 affect EGF receptor signaling, trafficking and sorting for degradation. We will investigate their effects on phosphorylation of EGFR, recruitment of adaptors, and downstream signaling as well as trafficking through the endosomal system, and sorting for down-regulation. These studies can be expected to provide insights into how normal cells function and the molecular basis for dysregulation of cell proliferation and migration associated with metastasis that occurs in cancer cells. Due to their crucial functions in regulating cell behavior these new molecules represent attractive targets for development of pharmacologic and anti-tumor agents.